Method for the suppression of viral growth

ABSTRACT

A method for suppressing undesired viral growth in a host which comprises administering to the host an effective amount of a compound of the formula: 
                 
 
wherein R 1 , R 2 , R 3  and R 4  are independently selected from the group consisting of HO—, CH 3 O— and CH 3 (C═O) O—. The method is exemplified by inhibiting Tat transactivation of a lentivirus and in suppressing Herpes simplex virus.

This is a continuation-in-part of U.S. application Ser. No. 08/627,588,filed Apr. 4, 1996 now U.S. Pat. No. 5,663,209, which is a division ofU.S. application Ser. No. 08/316,341, filed Sep. 30, 1994.

The invention described and claimed herein was made in part with fundsfrom Grant No. AI 32301 from the National Institutes of Health and inpart with funds from U.S. Army Medical Research Grant DAMD 17-93-C3122.The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the isolation, purification andcharacterization of derivatives of1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (nordihydroquaiareticnordihydroguaiaretic acid, NDGA) The derivatives were isolated from leafand flower extracts of the creosote bush (Larrea tridentata,Zygophyllaceae) and together with NDGA can be used to suppress Tattrans- activation in lentiviruses, including the HIV virus. Otherbroader aspects of the invention, including use of the disclosedcompounds for the treatment of Herpes simplex virus, will also behereinafter apparent.

2. Description of the Related Art

Tat is a transactivator of human immunodeficiency virus (HIV) geneexpression and is one of the two or more necessary viral regulatoryfactors (Tat and Rev) for HIV gene expression. Tat acts by binding tothe TAR RNA element and activating transcription from the long terminalrepeat (LTR) promoter.

The Tat protein stabilizes elongation of transcription and has also beenshown to be involved in transcription initiation. Previous studies haveshown that Tat mediates reduction of antibody-dependent T cellproliferation, contributing substantially to the failure of the immuneresponse. Tat also directly stimulates Kaposi's cell growth.

Since Tat has no apparent cellular homologs, this strong positiveregulator has become an attractive target for the development ofanti-AIDS drugs (see FIG. 1). In contrast to currently available HIVreverse transcriptase inhibitors (AZT, DDI) or potential proteaseinhibitors that prevent new rounds of infection, an inhibitor whichsuppresses viral gene Tat regulated expression of integrated proviralDNA will arrest the virus at an early stage (Hsu et al., Science254:1799-1802, 1991).

Efforts aimed at the elucidation of factors which control geneexpression at transcriptional and post-transcriptional levels in hosteukaryotes have recently made possible quantitative assessment of Tatfunction (Sim, Ann. N.Y. Acad. Sci. 616: 64-70, 1990, the entirecontents of which are hereby incorporated by reference and relied upon).To screen for inhibitors for Tat regulated transactivation (Tat-TRS),the secreted placental alkaline phosphatase (SEAP) reporter gene is putunder the control of HIV-1 LTR promoter in the plasmid pBC12HIV/SEAP.The Tat-coded activity is supplied by a second plasmid constructpBC12/CMV/t2. Transient cotransfection of COS cells with these twoplasmids leads to secretion of alkaline phosphatase into the culturemedium which is analyzed by a simple colorimetric assay (Berger et al.,Gene 66: 1-10, 1988, the entire contents of which are herebyincorporated by reference and relied upon) The SEAP assay, therefore,provides an indirect determination of Tat transactivation. An inhibitorshould cause reduction of the SEAP activity which is due to inhibitionof the expression of the SEAP mRNA via transactivation of the HIV-1 LTRpromoter by Tat protein (Tat-TRS).

SUMMARY OF THE INVENTION

In the present application, we disclose Tat-TRS inhibitory activity ofthe desert plant Larrea tridentata. Among several plant extractsprepared from rain forest and desert medicinal plants used intraditional medicinal against viral affections, only the total extractfrom the leaves and flowers of the creosote bush (Larrea tridentata)showed Tat-TRS inhibitory activity. This extract also inhibits HIVcytopathic effects on human lymphoblastoid cells chronically infectedwith the virus as assessed by the newly developed soluble-formazan assay(Weislow et al., JNCI 81: 577-586, 1989, the entire contents of whichare hereby incorporated by reference and relied upon).

The present invention discloses compounds of the structural formula:

wherein R₁, R₂, R₃ and R₄ are each selected from the group consisting ofHO—, CH₃O— and CH₃(C═O)O—, provided that R₁, R₂, R₃ and R₄ are not eachHO- simultaneously.

Each compound was isolated from leaf-flower extracts of the creosotebush Larrea tridentata and is a derivative of1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (nordihydroquaiareticnordihydroguaiaretic acid, NDGA).

In addition, NDGA and derivatives can be used to suppress Tattransactivation of a lentivirus, including the HIV virus, in a cell byadministering NDGA or a derivative thereof to the cell.

It is also contemplated according to the invention that the compoundsdisclosed herein can be used to inhibit Herpes simplex virus and otherundesired virus growth in a host as later described.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 graphically illustrates the life cycle of HIV-1 and differentsites of action of potential therapeutic agents including Tat-TRSinhibitors. The basal transcription step is indicated by 1 and the viralregulatory protein dependent transactivation step by 2.

FIG. 2 demonstrates the induction of the secreted alkaline phosphatase(SEAP) expression in the standard SEAP assay.

FIG. 3 shows the inhibition of Tat-TRS activity by creosote bush totalextract in the secreted alkaline phosphatase (SEAP) assay.

FIG. 4 shows an analysis of plant-derived HIV Tat inhibitors inComponent Lo by gas chromatography (GC) using an analyticalnon-destructive capillary cross-linked 5% phenyl-methylsiloxane (HP-5)column. Component Lo is a mixture of four components. The time (minutes)of elution appears on each peak.

FIG. 5 shows an analysis of plant-derived HIV Tat inhibitors inComponent Gr by gas chromatography (GC) using an analyticalnon-destructive capillary cross-linked 5% phenyl-methylsiloxane (HP-5)column. Component Gr is a complex mixture. The time (minutes) of elutionappears on each peak.

FIG. 6 illustrates the inhibition of Tat-induced SEAP expression by theplant-derived single compound Malachi 4:5-6 (Mal 4) and NDGA in thesecreted alkaline phosphatase (SEAP) assay.

FIG. 7 depicts the quantitation of XTT formazan production as ameasurement of viable cells in Mal 4 treated cultures of either HIVinfected or uninfected CEM-SS cells in two separate experiments. Thepercent XTT formazan production for infected cells without Mal 4treatment for experiment 1 was 8.9% and for experiment 2 was 7.5%. Forexperiment 1, uninfected cells are represented by • - - - • and infectedcells by x - - - x. In experiment 2, uninfected cells are represented by• - - - • and infected cells by x - - - x The EC₅₀ was found to be 4.25μg/ml or 13.4 μM. The IC₅₀ was estimated to be 100 μg/ml or 325 μM.

FIG. 8 is a photograph providing a comparative showing of HSV-1replication in a guinea pig which illustrates a further use of theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention discloses the isolation, purification andcharacterization of derivatives of1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane or nordihydroquaiareticnordihydroguaiaretic acid (NDGA). Each derivative of NDGA was isolated,purified and characterized according to the following procedures.

Materials and Methods

Cell line: COS-7 cells with SV40 origin of replication were maintainedin Isocove's Modified Dulbecco's Medium (IMDM) supplemented with lofetal calf serum (FCS) and antibiotics. Cells were incubated in ahumidified 95% O₂/5% CO₂ incubator at 37° C.

Plasmids: The plasmid pBC12/HIV/SEAP containing the Tat-sensitive HIVLTR promoter with the SEAP reporter gene but no Tat-coded function wasused to express SEAP basal activity; pBC12/CMV/t2 supplied the Tat-codedfunction, i.e., the induced SEAP level. pBC12/RSV/SEAP containing theconstitutive Tat-insensitive LTR promoter of Rous Sarcoma Virus (RSV)served as a positive control. All plasmids were obtained from Dr. BryanCullen, Duke Medical Center. Plasmids pSEAP and pBKCMV and HIV LTR andTat DNA are commercially available from Clontech and Stratagene. Plasmidtransformation was achieved in E. coli MC1061 strain, which was obtainedfrom Dr. Barbara Bachmann, Department of Biology, Yale University. E.coli MC1061 strain can also be purchased from Clontech. Plasmid DNAswere purified using Qiagen® purification kit (Qiagen).

Chemical Reagents: Diethanolamine (# 31589) and p-nitroph;enylphosphate(#71768) were purchased from Fluka BioChemika, and L-homoarginine(#H-1007) was purchased from Sigma Co. The lipospermine DOGS(Transfectam®, #E123 A, Promega) was used in DNA transfection studies.

Preparation of Plant Testing Materials: The leaves and flowers of thecreosote bush were collected based on ethnopharmacological inquiries.Plant materials were dried and ground in a 3 mm screen Willy mill. Inpilot studies, 1 g of the plant powder was initially extracted bysuccessive macerations using a mixture of chloroform:methanol. Theextract was concentrated to residue. The entire 176 mg of the crudeextract generated were treated 7 times with 3 ml of hexane. This stepafforded 137 mg of hexane-insoluble (HI) materials and 31 mg ofhexane-soluble (HS) materials. All these extracts were monitoredstepwise by SiO₂ TLC with cerium sulfate charring, 2% CeSO₄ (w/V) in5.6% H₂SO₄ (v/v), and by the SEAP assay for anti Tat-TRS activity.

For the SEAP assay, test materials were dissolved in 10% DMSO solutionmade in calcium/magnesium-free PBS. The suspension was centrifuged andthe stock solution (10 mg/ml) was filter-sterilized using a Millex®-GS22 μm filter (Millipore). Appropriate dilutions of the stock solutionwere prepared in a final DMSO concentration of 0.2% in PBS to obtain thevarious concentrations of test compounds.

Differential Practionation and Purification of the Active Ingredient byCountercurrent Chromatography (CCC): Based on the preliminary results ofthe above fractions in the SEAP assay, further fractionation of theactive HI fraction by CCC was undertaken. This led to the identificationof two major active fractions denoted “Green” and “Yellow” components.Identification of the most active fractions from these prospectivestudies prompted a full scale differential fractionation of plant powderin the attempt to generate a large quantity of the Green and Yellowfractions. This fractionation was carried out on 101.4 g of plantpowder, and started with a hexane treatment step, as outlined in Table1.

Further fractionation of the major components from the organic phase(OG) obtained after chloroform:water partition was achieved bycountercurrent liquid-liquid partition chromatography using theversatile cross-axis planet centrifuge (CPC) as described by Ito andConway, CRC Critical Reviews of Analytical Chemistry 17: 65 et seq.,1986, the entire contents of which are hereby incorporated by referencedand relied upon. The optimal solvent system was a mixture ofhexane:EtOAC:MeOH:0.5% NaCl in the ratio of 6:4:5:5, with the upperphase (organic layer) as the mobile phase. Five g of the organicfraction were dissolved in 23 ml of a mixture of the two phases andintroduced into the coil via a loop valve. The mobile phase pumpedthrough the coil while rotating it at 800 rpm. At a flow rate ofapproximately 4 ml per minute, approximately 32% of-the stationary phasewas initially lost (68%) retention). After the appearance of mobilephase in the elute, fractions of mobile phase were collected, evaporatedto dryness, monitored by TLC and pooled accordingly into 5 batches:Solvent front (SF), Green (Gr), Yellow (Ye), Red and Stationary phase(StP). All these fractions were then monitored by the SEAP assay foranti Tat-TRS activity.

Further purification of the Green and Yellow fractions was achieved byCCC using an epicyclic coil planet centrifuge known to the Itomultilayer coil separator-extractor (Ito and Conway, CRC CriticalReviews of Analytical Chemistry 17: 65 et seq., 1986, the entirecontents of which are hereby incorporated by reference and relied upon).The solvent system was a mixture of hexane:EtOAC:MeOH:0.5% NaCl(7:3:5:5).

Two hundreds mg of Green fraction afforded 6.8 mg of a component termedGr, i.e., =0.051% total yield based on the original plant powder.Similar studies on the Yellow fraction (Ye) generated 9.3 mg of acomponent denoted Lo. These purified components (Lo and Gr) eachconsists of several compounds, and their respective “mother” fractionswere stored at 4° C. until tested for biological activity and used forfurther characterization.

TABLE 1 DIFFERENTIAL FRACTIONATION AND COUNTERCURRENT CHROMATOGRAPHY(CCC) OF NDGA DERIVATIVES FROM THE CREOSOTE BUSH, LARREA TRIDENTATA.

Cell Culture and DNA Transfection: COS cells were maintained aspreviously described (Cullen, Cell 46: 973-982, 1986, the entirecontents of which are hereby incorporated by reference and relied upon).DNA transfection was performed using a modified procedure of thelipospermine (Transfectame, Promega #E123A) method originally describedelsewhere (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993,the entire contents of which are hereby incorporated by reference andrelied upon). Briefly, a day before DNA transfection, Linbro® 24 flatbottom well of 17-mm diameter plates were pretreated with 0.5 ml sterilesolution of 0.1% gelatin. The plates were kept in the hood for 1 hour(all transfection steps were performed in the hood, unless otherwisestated). The gelatin solution was aspirated and the plates were washedwith 0.5 ml of IMDM supplemented with 10% fetal calf serum andantibiotics (complete medium). COS cells were seeded at a density of≈1.5×10⁵ cells per 17-mm plate and incubated in a humidified 95% O₂/5%CO₂ incubator at 37° C. DNA transfection was performed at 30-50% cellconfluency. The stock solution of the Transfectam reagent, DOGS, wasprepared according to the manufacturer's advice at 1 mg/0.380 ml (2.38mg/ml or 3.4 mM) in 10% (v/v) ethanol in distilled water.

The transfection cocktail consisted of two solutions prepared in steriletubes:

-   -   a) Solution A contained a sterile 150 mM NaCl solution+plasmid        DNAs (non selected/selected gene in 2:1 ratio): 0.35 μg of        pBC12/HIV/SEAP per well+0.175 μg of pBC12/CMB/t2 (coding for Tat        function) per well.    -   b) Solution B Contained an equal volume of 150 mM NaCl and a        volume of Transfectame determined to be 6 times the total amount        of DNAS. Solutions A and B were homogenized and immediately        mixed.

Ten minutes were allowed for the reaction to proceed. Meanwhile, thegrowth medium was removed from the subconf luent COS cells and 300 μl(100 μl of complete IMDM+200 μl serum-free medium) were added to eachwell. The transfection cocktail was dispensed to the wells in equalvolume. Control samples containing no DNA were similarly treated andreceived sterile 150 mM NaCl solution alone. All samples were incubatedfor 10 and 12 hours after which 700 μl of complete growth medium wereadded. Test compounds prepared in 5% DMSO/Ca-Mg-free PBS (fornon-water-soluble materials) were immediately added at variousconcentrations to the wells. Drug-untreated control samples received 5%DMSO/PBS solution alone (final DMSO concentration of 0.2%). All sampleswere then incubated for an additional 48 hours after which 300 μl ofeach culture supernatant was removed for SEAP analysis.

The Secreted Alkaline Phosphatase (SEAP) Assay: The secreted alkalinephosphatase analysis was performed as originally described (Berger etal., Gene 66: 1-10, 1988, the entire contents of which are herebyincorporated by reference and relied upon). Briefly, a 250-μl aliquotwas removed from COS cell culture supernatants, heated at 65° C. for 5minutes to selectively inactivate endogenous phosphatase (SEAP is heatstable) and centrifuged in a microfuge for 2 minutes. One hundred μl of2×SEAP assay buffer (1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl₂; 10 mmL-homoarginine) were added to 100-μl aliquot of the samples. Thesolution was mixed and transferred into a 96-well flat-bottom culturedish (Corning). Twenty μl of pre-warmed substrate solution (120 mMp-nitrophenylphosphate dissolved in 1×SEAP assay buffer) were dispensedwith a multipipeter into each well containing the reaction mixture. A₄₀₅of the reaction was read at 5-minute intervals at 37° C. for 60 minutesusing an EL340i microplate reader (Bio-tek Instruments, Inc.) with5-second automatic shakings before each reading. The change inabsorbance was plotted against time in the standard assay of SEAPinduction. In the drug screening assay, the percent inhibition of SEAPexpression was calculated at 30 minutes as follows:% Inhibition=100−[(CT⁺−C⁺)×100]where:

C⁻: Control sample (no DNA, no drug)

CT⁻: Control sample (+DNA, no drug)

C⁺: Drug-treated sample (no DNA, +drug)

CT⁺: Drug-treated sample (+DNA, +drug)

Optimization of the Transfection Technique: Various techniques areutilized in the DNA transfection of eukaryotic cells. These proceduresinclude DNA coprecipitation with calcium phosphate or cationic polymers,cell membrane weakening either by chemical means (detergents, solvents,enzymes, amphophilid polymers) or by physical means (thermic, osmotic orelectric shocks, or particle bombardment). These techniques suffer, tosome extent, variable efficiency and varying degrees of cytotoxicity.

Prerequisites for cells to be amenable to DNA uptake, i.e., to cross theintact cytoplasmic membrane, are “compaction and masking of DNA charges”(Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). Theserequirements have been successfully met with the newly developedTransfectam® procedure. The Transfectam reagent (dioctadecylamidoglycylspermine) is a synthetic cationic lipopolyamine which contains apositively charged spermine headgroup with a strong affinity for DNA(K_(d)=10⁵−10⁻⁷ M). This spermine headgroup is covalently attached to alipid moiety by a peptide bond. The lipospermine molecules bind to DNA,coating it with a lipid layer. In the presence of excess lipospermine,cationic lipid-coated plasmid DNA vesicles are formed and the lipidportion of the complex fuses with cell membrane. DNA internalizationis-believed to occur by endocytosis.

Transfectam-mediated transfection has been shown to offer greaterefficiency than existing methods (Barthel et al., DNA and Cell Biology12 (6): 553-560, 1993). In addition, Transfectam® is a stable andvirtually non-cytotoxic reagent. However, factors for optimization oftransfection in the specific COS cell line had to be addressed. Thesefactors include the duration of transfection, the ratio of theTransfectam reagent to DNA, DNA concentration and other dilution factorssuch as NaCl volume and strength. The results of optimization oftransfection conditions are shown below.

a) Duration of transfection: COS cells were incubated with a fixedplasmid DNA concentration in time course studies. These studies aimed atthe selection of the suboptimal incubation time point for inhibitionstudies of SEAP expression by various test compounds. The results of thetime-course induction of SEAP expression (results not shown) indicate agradual time-dependent increase in SEAR expression. The onset of thisinduction began at less than 4 hours and reached a maximum at 24 hours.No significant difference was observed between the 10, 12 and 15-hourvalues. Therefore, the 12-15 hours endpoint was selected as theappropriate suboptimal incubation period for inhibition of SEAPexpression in all subsequent drug screen studies.

b) DNA concentration: The optimal DNA concentration for transfection wasdetermined based on previous studies with Transfectam reagent (Loefflerand Behr, Methods in Enzymology 217: 599-618, 1993). Forcontransfection, the ratio 2:1 (nonselected gene/selected gene) wasfound to be the most appropriate as reported elsewhere (Hsu et al.,Science 254: 1799-1802, 1991, the entire contents of which are herebyincorporated by reference and relied upon). The nonselectedpBC12/HIV/SEAP plasmid was utilized at a concentration of 0.35 μg/welland pBC12/CMV/t2 plasmic coding for Tat function at a concentration of0.75 μg/well in Linbro® 24 flat bottom well of 17-mm diameter plates.

c) Ratio of Transfectam to DNA and Determination of Ionic Strength: Theoptimal ratio of Transfectam® (DOGS) to plasmid DNA and the ionicstrength of NaCl used were a modification of the previously reportedvalues (Loeffler and Behr, Methods in Enzymology 217: 1799-1802, 1993)and determined as follows: From the original 1 mg/0.400 ml (2.38 mg/ml)stock solution of Transfectam® prepared in 10% (v/v) ethanol indistilled water, 6 times the volume (μl) of stock solution was requiredfor each μg DNA used. The optimal ionic strength of the solution wasprovided by an appropriate volume of 150 mM NaCl determined by therelation:Volume (μl) of NaCl=Volume (μl) Transfectam/0.6

The results of Tat-induced SEAP levels in the standard assay afteroptimization of these conditions, are illustrated in FIG. 2. Briefly,COS cells were maintained in Isocove's Modified Dulbecco's Medium (IMDM)supplemented with 10% fetal calf serum (FCS) and antibiotics. Triplicatecell samples were seeded at a density of ≈1.5×10⁵ cells per well inLinbro® 24 flat bottom wells of 17-mm diameter and incubated in ahumidified 95% O₂/5% CO₂ incubator at 37° C. until they reached 50%confluency. Subconfluent cells were transfected using the lipospermineprocedure (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993).The medium of the subconfluent cells was aspirated and replaced by 300μl of fresh minimum medium (IMDM supplemented with 3% FCS). COS cellswere transfected with either pBC12/HIV/SEAP alone (0.35 μg/well) orpBC12/CMV/t2 (coding for Tat function) at 0.175 μg/well+pBC12/HIV/SEAP(0.35 μg/well) or with buffer alone (no DNA control samples). The plateswere incubated for 12 to 15 hours after which, 700 μl of complete medium(IMDM containing 10% FCS) were added. Cells were then incubated for 48hours after which, a 250-μl aliquot was removed from COS cell culturesupernatants and heated at 65° C. for 5 minutes to selectivelyinactivate endogenous phosphatases (SEAP is heat stable). The sampleswere then centrifuged in a microfuge for 2 minutes. One hundred μl of2×SEAP assay buffer (1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl₂; 10 mML-homoarginine) were added to 100-μl aliquot of the samples. Thesolution was mixed and transferred into a 96-well flat-bottom culturedish (Corning). Twenty μl of pre-warmed substrate solution (120 mMp-nitrophenylphosphate dissolved in 1×SEAP assay buffer) were dispensedwith a multipipetter into each well containing the reaction mixture.A₄₀₅ of the reaction was read at 5-minute intervals at 37° C. for 60minutes using an EL340i microplate reader (Bio-tek Instruments, Inc.)with 5-seconds automatic shaking before each reading. The change inabsorbance was converted in mU of SEAP expression as previouslydescribed (Berger et al., Gene 66: 1-10, 1988) and plotted against time.

These results indicate a nearly 65-fold increase in SEAP induction after1 hour relative to the control (no DNA) levels or the induction ofnonselected gene (HIV/SEAP) alone.

Assay-Guided Isolation of Creosote Bush Extract Active Component(s) byCountercurrent Chromatography: As stated labove, the differentialfractionation and purification by countercurrent chromatography (CCC) ofthe creosote bush extract constituents led to the isolation of two majorcomponents (Table 1). 6.8 mg of the component termed Gr was isolatedfrom the Green fraction on SiO₂ TLC. The total percent yield was ≈0.051%based on the original plant powder. 9.3 mg of the component Lo wasisolated from the Yellow fraction (Ye).

Inhibition of Tat-TRS Activity by Extracts from Creosote Bush Leaves andFlowers: In several plant extracts tested with the SEAP assay, only theextract from the creosote bush, Larrea tridentata, leaves and flowersshowed significant inhibitory activity of HIV Tat protein. Creosote bushdisplayed a dose-response inhibition of SEAP expression as illustratedin FIG. 3. Briefly, triplicate samples of COS cells were transfectedwith a mixture of pBC12/HIV/SEAP and PBC12/CMV/t2 (coding for Tatfunction) in 2:1 ratio, using the lipospermine procedure as describedabove. Cells were incubated for 12-15 hours after transfection. Creosotebush extract stock solution (10 mg/ml) was made incalcium/magnesium-free PBS and 10% DMSO, and filter-sterilized using aMillex®-GS 22 μm filter (Millipore). The appropriate concentrations ofcreosote bush extract were added to the transfected cells at a finalDMSO concentration of 0.2% and samples were incubated for 48 hours. ForSEAP analysis, a 250-μl aliquot was removed from COS cell culturesupernatants, heated at 65° C. for 5 minutes to selectively inactivateendogenous phosphatases (SEAP is heat stable) and centrifuged in amicrofuge for 2 minutes. One hundred μl of 2×SEAP assay buffer (1.0 Mdiethanolamine, pH 9.8; 0.5 mM MgCl₂; 10 mM L-homoarginine) were addedto 100-μl aliquot of the samples. The solution was mixed and transferredinto a 96-well flat-bottom culture dish (Corning). Twenty μl ofpre-warmed substrate solution (120 mM p-nitrophenylphosphate dissolvedin 1×SEAP assay buffer) were dispensed with a multipipetter into eachwell containing the reaction mixture. A₄₀₅ of the reaction was read at5-minute intervals at 37° C. for 60 minutes using an EL340i microplatereader (Bio-tek Instruments, Inc.) with 5-second automatic shakingbefore each reading. The percent inhibition of SEAP expression wascalculated at 30 minutes as follows:% Inhibition=100−[(CT⁺−C⁺)/(CT⁻−C⁻)×100]where:

C⁻: Control sample (no DNA, no drug)

CT⁻: Control sample (+DNA, no drug)

C⁺: Drug-treated sample (no DNA, +drug)

CT⁺: Drug-treated sample (+DNA, +drug)

As seen in FIG. 3, the onset of this inhibition started at aconcentration of 20 μg/ml and reached a maximum inhibitory activity at600 μg/ml. The estimated EC₅₀ (the. concentration exhibiting 50% ofinhibition) for this crude material was 110 μg/ml. As the purificationof the active ingredients progressed, there was a stepwise increase inthe activity of the active ingredient(s) which tripled (68%) with theorganic phase (OG) fraction compared to 21% from the original totalcrude extract.

Inhibition of HIV cytopathic Effects: A compound inhibiting Tattransactivation should in principle block HIV replication. Consequently,creosote bush extract was tested at the National Cancer Institute (NCI)for inhibition of HIV-1 cytopathic effects using the soluble-formazanassay (Weislow et al., JNCI 81: 577-586, 1989, the entire contents ofwhich are hereby incorporated by reference and relied upon). Inprinciple, CEM-SS cells (ATCC, Rockville, Md.) are cocultivated withHIV-producing H9 cells. Viruses infect the host CEM-SS cells, replicateand kill most of the CEM-SS cells in a week. If the drug inhibits HIVproduction, CEM-SS cells are protected from HIV-induced cell death. Thetetrazolium (XTT) reagent is therefore metabolically reduced by theviable cells to yield a colored formazan product which is measurable bycolorimetry at 450 nm.

In practice, triplicate samples of CEM-SS cells (5000) were plated in96-well microtiter plate. Appropriate concentrations of test compoundswere added in a final volume of 100 μl calcium/magnesium-free PBS in 5%DMSO. Control samples received the compound medium (PBS) alone. Fiveminutes later, 500 highly infectious HIV-1 producing II9 cells or normalH9 cells were added to the wells containing the appropriate drugconcentrations. The microtiter plates were incubated at 37° C. in 95%O₂/5% CO₂ for 6 days after which a 50-μl mixture of XTT andN-methylphenazonium methosulfate (PMS) was added. The plates werereincubated for additional 4 hours for the color development (XTTformazan production). The plates were sealed, their contents were mixedby automatic shaking and the OD₄₅₀ of samples was determined in amicroplate reader. Each value represents the average of 3determinations. No significant difference was found between the means ofthe duplicate values of the uninfected cells and HIV-challenged cells,in presence of test compounds. In contrast, there was a significantdifference (p<0.05) between HIV-challenged samples in the presence orabsence of test compounds.

The results of these studies are summarized in Table 2. At aconcentration of 0.75 μg/ml for component Gr, there was an average 58%protection (cell viability) against HIV as opposed to 15% viability indrug-free samples challenged with HIV. At a concentration as low as0.187 μg/ml, component Lo exhibited even stronger inhibitory activity ofHIV cytopathic effects. The cell viability was 87%, very close to thatof not treated control cells (89%), in contrast to 14% viability for thedrug-free samples challenged with HIV. These compounds were devoid ofcytotoxicity at the concentrations used.

TABLE 2 INHIBITION OF HIV-1 CYTOPATHIC EFFECTS BY CREOSOTE BUSH EXTRACTCOMPOUNDS IN THE SOLUBLE-FORMAZAN ASSAY. Concentration Percent of livecells at of the test day 6 as measured by XXT sample which Formazanproduction yielded max Un- HIV HIV protection infected infected infectedagainst HIV plus plus minus without killing test test test Test Samplethe cells μ/ml sample sample sample Fraction Green 0.187 59 67 16Component G_(r) 0.75 80 67 16 -duplicates Component G_(r) 0.75 70 48 14Fraction 0.187 60 57 14 Yellow (Ye) Component L_(o) 0.187 91 86 14-duplicates Component L_(o) 0.187 89 87 14

Structure elucidation of the active components of creosote bush extract:The chemical characterization of the purified plant active constituentswas achieved mainly by mass spectroscopy and by H- and C- nuclearmagnetic resonance (NMR). Component Lo was found to be a mixture of fourrelated compounds (L1, L2, L3 and L4). Resolution and characterizationof each peak of the mixture was accomplished by gas chromatography (GC)using an analytical non-destructive capillary cross-linked 5%phenylmethylsiloxane (HP-5) column attached to a mass spectroscope (MS).The GC studies revealed that the first compound (L1) represented 6% ofthe mixture; the second (L2) was 76% of the mixture (MW=316); the third(L3) was isomeric with L2 and represented 9% of the total mixture(MW=316); and the fourth compound (L4) represented 9% of the mixture(MW=358). The time (minutes) of elution of these compounds is indicatedon the peaks (FIG. 4).

Component Gr consisted of fifteen compounds. Resolution andcharacterization of each peak of the mixture was accomplished by gaschromatography (GC) using analytical non-destructive capillarycross-linked 5% phenylmethylsiloxane (HP-5) column attached to a massspectroscope (MS). The GC studies revealed that four (G1, G2, G3 and G4)of the fifteen compounds. The time (minutes) of elution of thesecompounds is indicated on the peaks (FIG. 5).

The structures of these eight compounds (L1, L2, L3, L4, G1, G2, G3 andG4) are described as follows:

L1 has the composition C₁₈H₂₂O₄ and has been identified as a previouslyknown chemical, 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane(nordihydroquaiaretic nordihydroguaiaretic acid, NDGA, Merck Index, 10thEdition, 16534). The structural formula for L1 is as follows:

L2 has the composition C₁₉H₂₄O₄ and has been identified as3-O-methyl-NDGA or1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-hydroxyphenyl)-2,3-dimethylbutane.The structural formula for 3-O-methyl-NDGA is as follows:

L3 also has the composition C₁₉H₂₄O₄ and has been identified as4-O-methyl-NDGA or1-(3,4-dihydroxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-2,3-dimethylbutane.4-O-methyl-NDGA is also known as Malachi 4:5-6 or Mal 4. The structuralformula for 4-O-methyl-NDGA is as follows:

L4 has the composition C₂₁H₂₆O₅ and has been identified as3-O-methyl-4-O-acetyl-NDGA or1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane.The structural formula for 3-O-methyl-4-O-acetyl-NDGA is as follows:

G1 has a molecular weight of 344, a composition of C₂₁H₂₂O₄ and has beenidentified as 3,3′,4-tri-O-methyl-NDGA or1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane1-( 3 -methoxy- 4 -hydroxyphenyl)- 4 -( 3,4 -dimethoxyphenyl)- 2,3-dimethylbutane. 3,3′,4-tri-O-methyl-NDGA has the following structuralformula:

G2 has a molecular weight of 344, a composition of C₂₁H₂₂O₄ and has beenidentified as 3,4,4′-tri-O-methyl-NDGA or1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-diiaethoxyphenyl)-2,3-dimethylbutane1-( 3 -hydroxy- 4 -methoxyphenyl)- 4 -( 3,4 -dimethoxyphenyl)- 2,3-dimethylbutane. The structural formula for 3,4,4′-tri-O-methyl-NDGA isas follows:

G3 and G4 each has a molecular weight of 372 and a composition ofC₂₂H₂₈O₅. G3 is either 3′,4-di-O-methyl-3-O-acetyl-NDGA (as in G3a) or3,3′-di-O-methyl-4-O-acetyl-NDGA (as in G3b)

3′,4-di-O-methyl-3-O-acetyl-NDGA is also known as1-(3-methoxy-4-hydroxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylbutaneand has the following structural formula:

G3a:

3,3′-di-O-methyl-4-O-acetyl-NDGA is also known as1-(3-methoxy-4-hydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutaneand has the following structural formula:

Similarly, G4 is either 4,4′-di-O-methyl-3-O-acetyl-NDGA (as in G4a) or3,4′-di-O-methyl-4-O-acetyl-NDGA (as in G4b). 4,4′-di-O-3-O-acetyl-NDGA4,4′-di-O-methyl- 3 -O-acetyl-NDGA is also known as1-(3-hydroxy-4-methoxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylbutaneand has the following structural formula:

G4a:

3,4′-di-O-methyl-4-O-acetyl-NDGA is also known as1-(3-hydroxy-4-methoxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutaneand has the following structural formula:

G4b:

In addition to the above described isolation and purificationprocedures, each disclosed derivative of NDGA may be prepared bychemical synthesis following either methylation and/or acetylation ofNDGA according to the procedure of Ikeya et al., Chem. Pharm. Bull.27(7): 1583-1588, 1979, the entire contents of which are herebyincorporated by reference and relied upon.

Large scale purification of component Lo and two pure compounds (L2 andL3) from component Lo: A large scale CCC fractionation of a batch ofplant materials was initiated to generate a larger quantity of componentLo. A total of 110 g plant powder was first treated with 700 ml hexane 5times. The hexane soluble materials (1.17 g) were discarded. The hexaneinsoluble material (HI fraction) was dried and extracted 3 times bysuccessive macerations with 800 ml chloroform:methanol. This afforded 20g of total extract (Tex) which was combined with 7.6 g of HI fractionobtained from a previous batch differential extraction. A 27.6 g totalof crude plant materials thus generated was divided into two batches of10 g and 17.6 g. These batches were initially run separately on thelarge-capacity versatile Cross-Axis CPC (Shinomiya et al., J.Chromatogr. 644:215-229, 1993, the entire contents of which are herebyincorporated by reference) using the solvent systemhexane:EtOAc:MeOH:0.5% NaCl in 6:4:5:5 ratio with the upper phase(organic layer) as the mobile phase. The fractions were pooledexclusively according to the TLC patterns and four major fractions wereidentified from the two CCC operations and denoted fraction Green(Gr)(1.12 g), fraction Lo (2.87 g), fraction End (1.78 g) and finally,the stationary phase SP (20.84). The entire 2.87 g of fraction Lo wasfurther fractionated in the large model a triplet CPC with thehexane:EtOAc:MeOH:H₂O system in 7:3:5:5 ratio, using the aqueous layeras mobile phase. Four fractions denoted LYI (0.375); LYII (0.113 g);LYIII (0.280 g) and LYIV (2.80 g) were identified according to theelution order and the TLC patterns. These fractions were assayed foranti Tat-TRS activity at 10 μg/ml. Based on the test results, LYI wasselected for further purification.

Isolation of L2 and L3 pure compounds from LYI fraction of component Lo:The less hydrophobic fraction LYI was selected for further purificationusing the previously improved conditions, i.e., the triplet CPC andHex:CHCl₃:MeOH:10 mM NaCl solvent system in 1:4:4:2 ratio. This affordedpreparation of 148 mg of homogeneous L3 and 109.3 mg of pure L2 asexamined by NMR and mass spectroscopy. The structures of L3(Malachi4:5-6) and L2 have been described above.

Compounds L2 and L3 are derivatives of a previously identified chemical,1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethyl butane (nordihydroquaiareticnordihydroguaiaretic acid, NDGA, Merck Index, 10th Edition, #6534). Thestructural formula for NDGA, which is identical to that of L1 describedabove, is as follows:

The anti-HIV activity (the inhibition of Tat regulated HIVtransactivation) of NDGA and its derivatives was previously unknown.Comparative anti-HIV transactivation activity for NDGA and derivativeMalachi4:5-6 (Mal 4) are illustrated in FIG. 6. Briefly, duplicatesamples of subconfluent COS cells were co-transfected with plasmidpBC12/HIV/SEAP and pBC12/CMB/t2 (coding for Tat function) using thelipospermine procedure as described above. Cells were then incubated for12-15 hours. The test compounds were initially solubilized in 10%DMSOicalcium-magnesium-free DMSO in calcium-magnesium-free PBS and addedto the transfected cells in the appropriate concentrations at a finalDMSO concentration of 0.2%. The samples were incubated for 48 hoursafter which, a 250-μl aliquot was removed from COS cell culturesupernatants, and SEAP was analyzed as in the standard assay as in FIG.3. The percent inhibition of SEAP expression was calculated at 30minutes as follows:% Inhibition=100−[(CT⁺−C⁺)/(CT⁻−C⁻)×100]where:

C⁻: Control sample (no DNA, no drug)

CT⁻: Control sample (+DNA, no drug)

C⁺: Drug-treated sample (no DNA, +drug)

CT⁺: Drug-treated sample (+DNA, +drug)

Each point represents the average of two determinations. No significantdifference was apparent between the EC_(50s) of Mal 4 and NDGA whichwere 8 μg/ml (25 μM) and 6 μg/ml (20 μM), respectively. The EC_(50s) aredefined as the inhibitory concentration of the compound at which the Tatregulated HIV transactivation is reduced to 50% of that in untreatedcontrol cells.

The inhibition of transactivation of HIV promoter activity by Mal 4 andNDGA were compared in Table 3.

TABLE 3 INHIBITION OF TRANSACTIVATION OF HIV PROMOTER ACTIVITY BYNATURAL COMPOUNDS MAL 4 and NDGA. Inhibition of Tat-induced ScapExpression Test (% inhibition/concentration of test compound) Compound %μm % μm % μm % μm Mal 4 13.6 9.5 60.4 31.3 92 62.7 100 95 NDGA 17.0 9.973.8 32.6 88.1 65.2 92.9 99

The compounds NDGA and Mal 4 were assayed as described for FIG. 6.Control samples were run in quadruplicate. The percentage inhibition wasdetermined after 30 minutes and the OD₄₀₅ values were:

C⁻: Control sample (no DNA, no drug): 0.091

CT⁻: Control sample (+DNA, no drug): 0.805.

The quantitation of XTT formazan production as a measurement of viablecells in Mal 4 treated cultures of CEM-SS cells in shown in FIG. 7. Eachfigure shows infected or uninfected CEM-SS target cells (10⁴/M well)with serial dilutions of Mal 4. EC₅₀ represents the concentration of Mal4 (e.g. 13.4 μM) that increases (protects) XTT formazan production ininfected culture to 50% of that in uninfected, untreated culture cells.IC₅₀ represents inhibitory or toxic concentration of Mal 4 (e.g., 325μM, estimated) that reduces XTT formazan production in uninfectedcultures to 50% of that in untreated, uninfected control cells. Levelsof XTT formazan in untreated, infected control cells were 9% of those inuntreated, uninfected control cells. The soluble-formazan assay forHIV-1 cytopathic effects was conducted according to the proceduredescribed by Weislow et al., JNCI 81: 577-586, 1989, the entire contentsof which are hereby incorporated by reference and relied upon.

It will be appreciated that the invention in its broadest method aspectscontemplates the use of NDGA or any of its pharmaceutically acceptablederivatives for suppressing undesired viral growth in a host. Thesederivatives comprise modifications of NDGA where one or more, andpreferably all four, of the hydroxy groups are replaced by, for example,other substituents such as CH₃O—, CH₃(C═O)O—, or the like. Preferably,however, these substituents are selected to provide water-solubilitywithout affecting therapeutic properties. The active compounds to beused in the invention can, therefore, be structurally represented asfollows:

wherein R₁, R₂, R₃ and R₄ are each independent HO—, CH₃O—, CH₃(C═O)O— orother pharmaceutically acceptable equivalent thereof, with a preferencefor substituents which provide water-solubility. As representativewater-soluble derivatives of NDGA suitable for use herein, there may bementioned compounds of the foregoing formula wherein each R₁-R₄substituent is the group.

orthe group

Alternatively, the R₁ and R₂ substituents and the R₃ and R₄ substituentsmay be joined together with the adjacent carbon atoms of each phenylring to form cyclic structures as follows:

As indicated, the foregoing are given only for purposes of illustrationas those in the art will appreciate that other alternativepharmaceutically acceptable R₁-R₄ substituents may be employed toprovide water-solubility or to obtain functionally equivalentpharmacological results.

While the foregoing disclosure is directed primarily to the suppressionof HIV Tat transactivation, the compounds disclosed herein are alsoapplicable to suppress other undesired viral gene expression. In otherwords, the invention is not limited to use of the indicated compoundsfor the suppression of Tat transactivation of a lentivirus. Theeffectiveness of the compounds against other viral gene expression canbe readily determined by testing the compounds in appropriateart-recognized test systems. Thus, for example, the compounds of theinvention have been found to be effective for inhibition of the Herpessimplex virus. Without intending to be bound by any theory as to how thecompounds function against this virus or other virus, it is believedthat the compounds proceed by a common mechanism which includesinhibiting proviral transcription and transactivation essentially asearlier described herein.

This broader application of the invention is illustrated hereafter withdata showing the suppression of Herpes simplex virus replication byinhibition of transcription and transactivation of HSV IE gene α₄(ICP₄).

By way of explaining this further aspect of the invention, it isgenerally known that manifestation of Herpes simplex virus (HSV-1,HSV-2) infection has frequently been observed in HIV infected patients.Initial treatments with anti viral agents (such as acyclovir for HSV andreverse transcriptase and viral protease inhibitors for HIV-1) have beenfound to be quite effective. However, drug resistance and highsensitivity to multiple drugs often developed in these patients withtime. Antiviral strategies aimed at controlling the replication of wildtype and mutants of both viruses by drugs which can control both virusesthus are clinically important. Transcription of proviral HIV and HSV IEgenes (α₀, α₄, α₂₂, α₂₇, and α₄₇) (Ref. Deluca, N. A. and Schaft, P., J.Virol. 14, 8, 1974) are essential for replication of these tworespective viruses. The expressions are thoroughly host dependent,utilizing RNA polymerase II and cellular transcription factors. Thepromoter activities of proviral HIV and HSV α₄ (ICP₄) gene are regulatedby host protein Spl in addition to other cellular factors (Ref. Jones,K. A. and Tjian, R., Nature 317, 179 1985).

The use of the invention for suppressing Herpes simplex virus (HSV-1,HSV-2) is illustrated hereinafter using tetramethoxylnordihydroguaiaretic acid (CH₃—O)₄ NDGA, 4N of the formula:

to inhibit HSV ICP₄ gene transactivation and HSV replication in verocells at a dosage range that is not toxic to the host cells (see Table4).

In addition, in comparison with acyclovir (ACV), a commerciallyavailable HSV drug, HSV virus showed no drug resistance towardtetramethoxyl NDGA (4N) following ten viral passages in vero cells. TheIC₅₀ for 4N remained essentially the same from first passage to tenthpassage (IC₅₀ app 10 μm) while the concentration of acyclovir requiredto inhibit HSV-1 increased from 7.5 μm at first passage to 440 μm at the10th passage (see Table 5A) Consequently, tetramethoxyl NDGA, 4N, wasable to also inhibit the acyclovir resistant strain (HSV-1 SM44-CV4r)very effectively (IC₅₀ of 6.9 μm) (see Table 5B).

It is also noted that tetramethoxyl NDGA 4N has been used in an animalsystem, i.e. to treat HSV infected skin of guinea pigs. At aconcentration of 60 mg/ml, 4N eliminated HSV-1 growth completely in fivedays following daily topical treatment of the drug at the infected skinareas (FIG. 8).

Two male guinea pigs (350 gm body weight) were used in the guinea pigexperiment. The back skin of the animal was shaved and equally dividedinto six patches. Each skin patch was pinched with seven-pin needles andHSV-1 suspension was applied topically to infect each punched area.Twenty-four hours after infection, three agents (ABPS, 2 mg/ml, ABPS 4mg/ml, tetramethoxyl NDGA, 4N 60 mg/ml and acyclovir 60 mg/ml) wereapplied to the infected areas five times per day for five days. Picture(FIG. 8) was taken 96 hours following infection.

Comparing with the two positive controls (HSV-SC, HSV-C, i.e. infectionwithout drug treatment), the result indicated that agent ABPS isineffective while tetramethoxyl NDGA and acyclovir are effective insuppression of HSV-1 replication. Differing from acyclovir, however,tetramethoxyl NDGA has the advantage in that it is a mutationinsensitive drug (Table 5A and Table 5B).

TABLE 4 Tetramethoxyl NDGA Effect Concentration of (CH₃O)₄ NDGA μM %Inhibition IC₅₀ I. Inhibition of HSV-1 Replication in Vero Cells 1497.50 7.7 μM 7 40 3.5 20 1.75 <0 0.875 <0 0.437 <0 II. Inhibition ofHSV-1 ICP4 Transactivation in Vero Cells 80 78 33 μM 70 68 60 64 50 5240 35 30 30 20 8 III. Inhibition of Growth of Vero Cells 280 79.62 142μM 140 43.95 70 9.55 35 7.64 17.5 7.64 8.75 3.18 4.37 1.27

TABLE 5 Comparison Between the Drug Sensitivities of Acyclovir andTetramethoxyl NDGA Against HSV-1 in Vero Cells A. IC₅₀ at differentpassages IC50 μM Passage ACV [CH₃]₄ NDGA 1 7.5 11.7 2 37.8 4.4 3 >88.88.2 4 138.4 5.9 5 >220 9.9 10 440 10Virus which recovered from medium of each passage at IC₅₀ drugconcentration were used to infect the subsequent passage of the cells.

B. Drug sensitivities of tetramethoxyl NDGA against ACV sensitive or ACVresistant strain of HSV-1 HSV-Sm44-ACVg HSV-18m44-CV4r Drug (IC50) (μM)(IC50) ACV <6.9 117.6 [CH₃]₄ NDGA 6.9 6.9

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not to be limited to thedisclosed embodiment, but on the contrary is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

Thus, it is to be understood that variations in the derivatives of NDGAand the method of suppression of Tat transactivation can be made withoutdeparting from the novel aspects of this invention as defined in theclaims.

1. A method for suppressing viral growth in a host which consistsessentially of administering to the host an effective viral growthsuppressing amount of a composition consisting essentially of a compoundof the formula:

wherein R₁, R₂, R₃ and R₄ are each selected from the group consisting ofHO—, CH₃O— and CH₃(C═O)O—, provided that R₁, R₂, R₃ and R₄ are not eachHO—.
 2. The method of claim 1 4, wherein said compound is thewater-soluble substituent is —O(C═O)CH ₂ NH(CH ₃)₂ .Cl.
 3. The method ofclaim 1 for suppressing 4, wherein the host is infected with Herpessimplex virus in the host .
 4. A method for suppressing viral growth ina host infected with a virus comprising (a) providing a compositioncomprising a substantially purified compound and (b) administering saidcomposition to the host in a dosage having an effective amount of thecompound to suppress viral growth, wherein the compound is a derivativeof nordihydroguaiaretic acid (NDGA) having the formula:

wherein R ₁ , R ₂ , R ₃ and R ₄ are each selected from the groupconsisting of HO—, CH ₃ O— and CH ₃(C═)O—, or a water solublesubstituent, provided that R ₁ , R ₂ , R ₃ and R ₄ are not each HO—,wherein the water soluble substituent is selected from the groupconsisting of: —O(C═O)CH ₂ NH(CH ₃)₂ .Cl, —O(C═O)CH ₂ NH ₂,


5. The method of claim 4, wherein the water-soluble substituent is—O(C═O)CH ₂ NH ₂ .
 6. The method of claim 4, wherein the compoundinhibits viral transcription.
 7. The method of claim 4, wherein thecompound inhibits transactivation of viral gene.
 8. The method of claim4, wherein the compound is 1-( 3,4 -dihydroxyphenyl)- 4 -( 3 -hydroxy- 4-methoxyphenyl)- 2,3 -dimethylbutane ( 4 -O-methyl-NDGA).
 9. The methodof claim 4, wherein the compound is 1-( 3,4 -dihydroxyphenyl)- 4 -( 3-methoxy- 4 -acetoxyphenyl)- 2,3 -dimethylbutane ( 3 -O-methyl- 4-O-acetyl-NDGA).
 10. The method of claim 4, wherein the compound is 1-(3 -methoxy- 4 -hydroxyphenyl)- 4 -( 3,4 -dimethoxyphenyl)- 2,3-dimethylbutane ( 3,3′,4 -tri-O-methyl-NDGA).
 11. The method of claim 4,wherein the compound is 1-( 3 -hydroxy- 4 -methoxyphenyl)- 4 -( 3,4-dimethoxyphenyl)- 2,3 -dimethylbutane ( 3,4,4′-tri-O-methyl-NDGA). 12.The method of claim 4, wherein the compound is 1-( 3 -methoxy- 4-hydroxyphenyl)- 4 -( 3 -acetoxy- 4 -methoxyphenyl)- 2,3 -dimethylbutane( 3′,4 -di-O-methyl- 3 -O-acetyl-NDGA).
 13. The method of claim 4,wherein the compound is 1-( 3 -methoxy- 4 -hydroxyphenyl)- 4 -( 3-methoxy- 4 -acetoxyphenyl)- 2,3 -dimethylbutane ( 3,3′-di-O-methyl- 4-O-acetyl-NDGA).
 14. The method of claim 4, wherein the compound is 1-(3 -hydroxy- 4 -methoxyphenyl)- 4 -( 3 -acetoxy- 4 -methoxyphenyl)- 2,3-dimethylbutane ( 4,4′-di-O-methyl- 3 -O-acetyl-NDGA).
 15. The method ofclaim 4, wherein the compound is 1-( 3 -hydroxy- 4 -methoxyphenyl)- 4 -(3 -methoxy- 4 -acetoxyphenyl)- 2,3 -dimethylbutane ( 3,4′-di-O-methyl- 4-O-acetyl-NDGA).
 16. The method of claim 4, wherein R₁ , R ₂ , R ₃ and R₄ are not each CH ₃ O— or CH ₃(C═O)O— simultaneously.
 17. The method ofclaim 4, wherein the effective viral growth suppressing amount of thecompound is less than 95 μM.
 18. The method of claim 4, wherein theeffective viral growth suppressing amount of the compound is less than62.7 μM.
 19. The method of claim 4, wherein the effective viral growthsuppressing amount of the compound is less than 31.3 μM.
 20. The methodof claim 4, wherein the effective viral growth suppressing amount of thecompound is less than 25 μM.
 21. The method of claim 4, wherein theeffective viral growth suppressing amount of the compound is less than9.5 μM.
 22. A method of inhibiting replication of an acyclovir-resistantvirus in a cell comprising the steps of: (a) providing a substantiallypurified compound having a formula:

wherein R ₁ , R ₂ , R ₃ and R ₄ are each selected from the groupconsisting of HO—, CH ₃ O— and CH ₃(C═O)O—, and a water solublesubstituent, wherein the water soluble substituent is selected from thegroup consisting of: —O(C═O)CH ₂ NH(CH ₃)₂ .Cl, —O(C═O)CH ₂ NH ₂,

(b) contacting the cell with the compound.
 23. A method of treatment ofacyclovir-resistant viral infection in a subject comprising the stepsof: (a) providing a substantially purified compound having the formula:

wherein R ₁ , R ₂ , R ₃ and R ₄ are each selected from the groupconsisting of HO—, CH ₃ O— and CH ₃(C═O)O—, and a water solublesubstituent, wherein the water soluble substituent is selected from thegroup consisting of: —O(C═O)CH ₂ NH(CH ₃)₂ .Cl, —O(C═O)CH ₂ NH ₂,

(b) administering the substantially purified compound to the subject.24. A method of treatment of a subject infected with a virus, whereinthe virus is resistant to acyclovir comprising the steps of: (a)providing a composition comprising a substantially purified compound;and (b) administering said composition in a dosage having atherapeutically effective amount of the compound to the subject, whereinthe compound has the formula:

wherein R ₁ , R ₂ , R ₃ and R ₄ are each selected from the groupconsisting of HO—, CH ₃ O— and CH ₃(C═O)O—, and a water solublesubstituent, wherein the water soluble substituent is selected from thegroup consisting of: —O(C═O)CH ₂ NH(CH ₃)₂ .Cl, —O(C═O)CH ₂ NH ₂,


25. The method of claim 24, wherein the water-soluble substituent is—O(C═O)CH ₂ NH ₂ .
 26. The method of claim 24, wherein the water-solublesubstituent is —O(C═O)CH ₂ NH(CH ₃)₂ .Cl.
 27. The method of claim 24,wherein the compound inhibits viral transcription.
 28. The method ofclaim 24, wherein the compound inhibits transactivation of the viralgene.
 29. The method of claim 24, wherein the compound is 1-( 3,4-dihydroxyphenyl)- 4 -( 3 -hydroxy- 4 -methoxyphenyl)- 2,3-dimethylbutane ( 4 -O-methyl-NDGA).
 30. The method of claim 24, whereinthe compound is 1-( 3,4 -dihydroxyphenyl)- 4 -( 3 -methoxy- 4-acetoxyphenyl)- 2,3 -dimethylbutane ( 3 -O-methyl- 4 -O-acetyl-NDGA).31. The method of claim 24, wherein the compound is 1-( 3 -methoxy- 4-hydroxyphenyl)- 4 -( 3,4 -dimethoxyphenyl)- 2,3 -dimethylbutane (3,3′,4 -tri-O-methyl-NDGA).
 32. The method of claim 24, wherein thecompound is 1-( 3 -hydroxy- 4 -methoxyphenyl)- 4 -( 3,4-dimethoxyphenyl)- 2,3 -dimethylbutane ( 3,4,4′-tri-O-methyl-NDGA). 33.The method of claim 24, wherein the compound is 1-( 3 -methoxy- 4-hydroxyphenyl)- 4 -( 3 -acetoxy- 4 -methoxyphenyl)- 2,3 -dimethylbutane( 3′,4 -di-O-methyl- 3 -O-acetyl-NDGA).
 34. The method of claim 24,wherein the compound is 1-( 3 -methoxy- 4 -hydroxyphenyl)- 4 -( 3-methoxy- 4 -acetoxyphenyl)- 2,3 -dimethylbutane ( 3,3′-di-O-methyl- 4-O-acetyl-NDGA).
 35. The method of claim 24, wherein the compound is 1-(3 -hydroxy- 4 -methoxyphenyl)- 4 -( 3 -acetoxy- 4 -methoxyphenyl)- 2,3-dimethylbutane ( 4,4′-di-O-methyl- 3 -O-acetyl-NDGA).
 36. The method ofclaim 24, wherein the compound is 1-( 3 -hydroxy- 4 -methoxyphenyl)- 4-( 3 -methoxy- 4 -acetoxyphenyl)- 2,3 -dimethylbutane (3,4′-di-O-methyl- 4 -O-acetyl-NDGA).
 37. A method of treatment of viralinfection in a host comprising the steps of: (a) providing a compositioncomprising a compound; and (b) administering said composition in adosage having a viral inhibitory amount of the compound to the host,wherein the compound has the formula selected from the group consistingof: